Qualitative Determination of Indole Alkaloids of Tabernaemontana fuchsiaefolia ( Apocynaceae )

Marcos A. Zocoler, Arildo J. B. de Oliveira, Maria H. Sarragiotto, Viviane L. Grzesiuk and Gentil J. Vidotti* a Departamento de Fármacos e Medicamentos, Universidade do Oeste Paulista, Rua José Bongiovani 1297, 19050-680 Presidente Prudente SP, Brazil b Departamento de Farmácia e Farmacologia, and c Departamento de Química, Universidade Estadual de Maringá, Avenida Colombo 5790, 87020-900 Maringá PR, Brazil


Introduction
The genus Tabernaemontana belongs to the Apocynaceae family.Several species of this genus are used in folk medicine against many diseases: diarrhoea, skin affections, warts, syphilis, Hansen's Disease, cancer and insect bites. 1 The main constituents of the Tabernaemontana genus are indole alkaloids, a class of substances with a wide range of pharmacological activities: cholinesterase inhibitors, 2 analgesic, anti-inflammatory, bactericidal, oestrogenic, and stimulant and depressant of the central nervous system (CNS). 3Taxonomy of the Apocynaceae family is quite complex and there are difficulties in its classification and terminology because there are a great number of synonyms.Both generic names, Tabernaemontana and Peschiera, have been used in Brazil and are matter of discussion. 4 Tabernaemontana fuchsiaefolia A. DC., known as "leiteira" (milk weed), is a common lactiferous tree that grows in pastures of the Brazilian States of São Paulo and northern Paraná. 5,6In Brazil, this species is commonly used to treat malaria. 7ederici et al. 8 reported that the extracts of Tabernaemontana fuchsiaefolia stem and root barks have good in vitro activity against Plasmodium falciparum, the parasite responsible for malaria. 8Casado et al. 9 showed that the crude extract obtained from Tabernaemontana fuchsiaefolia stem barks presented depressant activity in CNS and also provoked spontaneous stimulation in isolated rat womb. 9ne of the problems related to the use of vegetal crude extracts is the variation of concentration of their active ingredient due to crop time, place and soil properties.][12][13][14] In the present study, we have used GC-MS to effect the analyses of alkaloid extracts from different parts of Tabernaemontana fuchsiaefolia.

Plant material
Leaves, and stems and roots of Tabernaemontana fuchsiaefolia were collected on the Universidade Estadual de Maringá (UEM) campus in November 1999 and identified by Dr. Maria Conceição de Souza.A specimen of Tabernaemontana fuchsiaefolia (HUM no.Reg.8,325) was deposited in the Biology Department herbarium of UEM.
Table 2 shows the indole alkaloid contents in alkaloidal extracts obtained from various parts of T. fuchsiaefolia and some literature data.The GC-MS chromatographic profile of these fractions shows that the stem bark extracts have the largest number and the largest concentrations of alkaloids.The corynanthean alkaloid class (akuammidine-group and vobasine-group) was not detected in the leaves.Only voachalotine, an alkaloid belonging to the corynanthean class (akuammidinegroup), was detected, but in low concentration, in root bark extracts.Two alkaloids from this same class, 16-epiaffinine (the vobasine-group) and affinisine (akuammidine-group), were detected in stem bark extracts in higher concentration.Alkaloids with the ibogan skeleton, such as coronaridine, voacangine, ibogamine, and tabernanthine were present in all extracts analyzed, of which voacangine was the most abundant one.Coronaridine was more abundant in roots than in other parts analyzed.According to Delorenzi, 23 coronaridine, which is the main indole alkaloid, of the chloroform fraction obtained from the ethanolic bark extract of Peschiera australis, is responsible for its action against Leishmania amazonensis.The indole alkaloid voachalotine, which is the chemosystematic marker of this species¹, was detected in stem and root bark extracts.

Conclusions
Gas chromatography coupled to mass spectrometry (GC-MS) proved to be a valuable tool for the analysis of T. fuchsiaefolia monomeric indole alkaloids.A total of fifteen indole alkaloids could be quickly and easily identified in alkaloidal extracts from different parts of T. fuchsiaefolia.GC-MS retention time and standard addition provided the identity of five indole alkaloids present in the extracts.Mass spectrometry analysis confirmed the presence of alkaloids coronaridine, voacangine, voachalotine, 16-epi-affinine, and affinisine, and indicated the presence of ten other alkaloids.However, this method is limited to the analysis of less polar components, unless a derivation of the most polar components is provided. 14erivine, vobasine, vobasinol, heyneanine, voacristine, and 3-hydroxycoronaridine previously reported in T. fucshiaefolia, were not detected in this study probably due to their low concentration at collecting time or the isolation method used.We have used the neutral method (ethanol) to prepare the extracts for GC-MS analysis, while the alkaloids 8,24,25 reported were obtained by either acid or basic methods.

Table 1 .
CG-MS experimental data, retention time, M +• and main fragments m/z (relative intensity) for indole alkaloids of F1 fraction from T. fuchsiaefolia